A New Enrichment Method for the Selective Isolation of Streptomycetes from the Root Surfaces of Herbaceous Plants

Actinomycetologica (2007) 21:66–69 Copyright 2007 The Society for Actinomycetes Japan VOL. 21, NO. 2 NOTE A New Enrichment Method for the Selective Isolation of Streptomycetes from the Root Surfaces of Herbaceous Plants

Emi Matsukawa, Youji Nakagawa, Yuzuru Iimura, and Masayuki Hayakawa Division of Applied Biological Sciences, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Takeda-4, Kofu 400, Japan (Received Jun. 8, 2007 / Accepted Oct. 26, 2007 / Published Nov. 30, 2007)

Diverse rhizoplane streptomycetes with high levels of anti-phytopathogenic activity were eﬃciently isolated from healthy herbaceous plants using a new enrichment method, which we designated as the moist incubation and desiccation (MI&D) method. The MI&D method involves incubating root tissues on humic acid-vitamin (HV) agar, desiccating roots bearing arthrospore chains of colonized streptomycetes in dried soil particles, then liberating spores by agitation in water. Dilutions of the liquid enriched with streptomycete spores are then plated and incubated on HV agar. The desiccation stage drastically reduces bacterial contamination, thereby achieving selective isolation of streptomycetes.

Species of the genus Streptomyces produce a wide colonies are repeatedly transferred onto fresh agar medium variety of antibiotics and continue to be a major source of or transferred onto a membrane filter on the same plate.7,10) useful secondary metabolites.1) The isolation and subse- Although these isolation methods have yielded many quent screening of Streptomyces spp. from diverse habitats streptomycete subcultures, the purification stage tends to is important for the identification of useful strains that be laborious and time-consuming. produce novel bioactive compounds. Although most Strep- In the present paper we describe a new enrichment tomyces spp. inhabit soils and are saprophytic, digesting method that is effective for the selective isolation of animal and plant remains,2) some species have intimate diverse streptomycetes from healthy plants. The method associations with living plants.3) Pathological interactions utilizes the superior ability of streptomycete spores to between plants and the endophytic streptomycetes Strepto- withstand desiccation. We also report the antibiotic activity myces scabies, S. acidiscabies, and S. turgidiscabies have of streptomycete isolates against several phytopathogenic been reported.3,4) In recent years, a range of streptomycetes microorganisms. and non-streptomycetes have been isolated from both surface-sterilized and untreated leaves, stems and roots Effects of pretreatments on isolation of rhizoplane from healthy plants.5–10) New bioactive compounds, such streptomycetes as alnumycin,11) fistupyrone12) and the munumbicins,13) Roots (1–3 mm in diameter) were sampled from nine have been found to be produced by these plant-associating healthy herbaceous plants, each a different species, growing streptomycetes. Some endophytic streptomycetes can also in agricultural fields in Nagano, Shizuoka and Yamanashi be used as biological control agents against plant patho- prefectures, Japan. The roots were used for actinomycete gens.9) isolation within 24 h. The root samples were first washed in Several methods have been used for isolating strepto- running tap water to remove soil particles, then cut into mycetes and other actinomycetes from living plants. For pieces of 5–10 mm in length. Fifty milligrams of each root example, Hunter et al.5) isolated epiphytic streptomycetes sample was placed into 10 ml sterile tap water in a test by momentarily touching fresh pieces of leaves, stems and tube and vigorously stirred for 60 sec using a thermomixer roots from various species of plants to the surfaces of plates (Thermonics Co., Ltd., Tokyo). The washing process was containing an agar medium and incubating the plates. repeated using a fresh 10 ml sterile tap water, then the Several workers have isolated streptomycetes from pieces root sample was rinsed in sterile tap water containing of healthy plant tissue by incubating the samples on an cycloheximide (100 mg/ml) and nystatin (100 mg/ml) for appropriate agar medium prior to isolation.6–10) Previous 60 sec. The rinsed root sample was transferred temporarily attempts at isolating endophytic species have involved onto sterile filter paper to eliminate excess moisture and sterilization of plant tissue surfaces in ethanol, sodium then placed on a plate of HV agar14) supplemented with hypochlorite or propylene oxide.10) Streptomycete colonies cycloheximide (50 mg/ml), nystatin (50 mg/ml), nalidixic can be seen easily on the surfaces of incubated plant acid (20 mg/ml) and trimethoprim (20 mg/ml). The plate tissues, but they are usually associated with bacterial was incubated for two weeks at 30C. Each root sample and fungal colonies. For purification, these streptomycete colonized by streptomycetes was then removed from the

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Table 1. Effects of moist incubation (MI) and subsequent desiccation of Perilla frutescens roots on isolation of actinomycetes Colony-forming units§ per g of plant root sample Other Non-filamentous Streptomycetes Treatment Streptomycetes actinomycetes bacteria % None (control) 0:8 ð0:2Þ103a Not detected 2:5 ð1:5Þ105a 3.2 Moist incubationy 2:7 ð0:1Þ108b 2:5 ð0:2Þ106a 7:5 ð0:8Þ108b 26.4 MI and desiccationz 1:5 ð0:1Þ108b 2:2 ð0:2Þ106a 4:0 ð2:1Þ107c 78.0 Washed root samples were used. yOn HV agar for two weeks at 30C. zIn dried fine soil particles for 24 h at 30C. §Counts represent the means of triplicate experiments (triplicate plate counts for each experiment) with standard deviations given in parentheses. Within each column, means with the same superscript are not significantly different (p < 0:05) according to Duncan’s multiple range test. plate surface and suspended in 1.5 g sterile, dry, fine soil the genera Actinoplanes, Micromonospora and Nocardia. particles that had been earlier placed in a test tube. The Although this protocol also concomitantly increased bacte- sterile soil particles were prepared as previously describ- rial contamination, further treatment of the incubated root ed.15) To ensure low humidity in the test tube, dried silica samples in dried soil particles resulted in a drastic reduction gel granules were wrapped in sterile filter paper and used as in the number of unicellular bacteria. This desiccation a plug for the tube. The tube was vigorously stirred using a stage had no adverse effect on the number and diversity of thermomixer and subsequently kept in a glass desiccator streptomycetes recovered. Thus, the integrated procedure with a sufficient amount of dried silica gel at 30C for 24 h. consisting of initial incubation of root samples, desiccation Ten milliliters of sterile tap water was then poured into the of the colonized roots in dried soil particles, and plating tube containing the soil particles and root tissues, and the onto HV agar, yielded a high colony count of strepto- mixture was vigorously stirred using a thermomixer for mycetes that accounted for 78% of the total colonies 60 sec. The tube was allowed to stand for 60 sec to allow recovered. Figure 1a shows the formation of discrete and the coarse sand to precipitate, then the supernatant was powdery actinomycete colonies from a root fragment of diluted with sterile tap water (1:1,000) and aliquots (0.2 ml) P. frutescens on HV agar. Scanning electron microscopy were plated in triplicate on HV agar supplemented with confirmed the formation of spiral or straight arthrospore the same antibiotics as described above. The plates were chains from the surface of the root fragment, which typify incubated at 30C for 14 d. As a control experiment, a the genus Streptomyces (Fig. 1b). washed root sample was directly suspended in soil particles The efficiency of the new enrichment method, which we that had been dampened previously using 1 ml sterile tap designate the moist incubation and desiccation (MI&D) water. The mixture was stirred and 9 ml sterile tap water method, for isolating streptomycetes was confirmed by was added to the soil particles. The mixture was stirred applying it to root samples from an additional eight again and portions of the supernatant were plated out in the herbaceous plants (Brassica oleracea, Lupinus mutabilis, same way. All experiments were performed in triplicate. Mentha piperrita, Mentha suaveolens, Pisum sativum, Actinomycetes and unicellular bacteria appearing on the Solanum lycopersicum, Spinacia oleracea and Tagetes plates were identified by eye and with the aid of a light erecta). The MI&D method selectively isolated diverse microscope. Streptomyces strains were identified as those streptomycetes from all samples, with streptomycetes isolates forming discrete and floccose, powdery, or velvety constituting 3–52% of the total bacterial population colonies. Microscopy subsequently confirmed the forma- recovered. tion of branching, stable substrate hyphae, and aerial hyphae with long spore chains that could be categorized as Characterization of streptomycete isolates rectiflexibiles, retinaculiaperti or spirales spore chains on A total of 71 rhizoplane Streptomyces strains, isolated the basis of their characteristic morphology.2) from nine herbaceous plant species using the MI&D Pretreatment techniques and HV agar supplemented method described above, were selected at random and with antifungal and antibacterial antibiotics were used to subcultured on oatmeal-YGG slants.16) In addition, 41 selectively isolate streptomycetes from the roots of Perilla Streptomyces strains, isolated on HV plates from the soils frutescens (Table 1). Streptomycetes were recovered from in which these plants were grown, were simultaneously washed root tissues of P. frutescens, but in low num- subcultured. In order to validate genus-level identification bers. We therefore attempted to increase the quantity of using light microscopy, these isolates were analyzed for streptomycetes recovered. Initial incubation of the root cell wall DAP configuration using the rapid method of samples on HV agar significantly increased the recovery of Hasegawa et al.17) Isolates were confirmed to contain L- streptomycetes as well as of other actinomycetes, such as DAP, which is typical for Streptomyces.

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Table 2. Phenotypic characterization of streptomycetes isolated A from the root surface of Perilla frutescens and from soil sampled at a location 2 m distant from the growing plant No. of strains Spore Substrate Spore Melanin Root Soil color color chains pigment surface Gray Orange RF 10 Gray Orange S 01 Gray Green RF 01 Gray Yellow-brown S 14 Gray Yellow-brown RF 01 Green Yellow-brown S 01 Red Red-orange RA 02 B Red Red-orange RF 03 Red Yellow-brown RF 01 Yellow Yellow-brown S 01 Yellow Hyaline RF 10 White Yellow-brown RF 01 Blue Blue S + 1 1 Gray Yellow-brown RA + 1 0 Gray Yellow-brown RF + 4 1 Gray Yellow-brown S + 6 0 10 µm Gray Red-orange S + 1 0 White Red-orange RF + 1 0 Fig. 1. Growth of streptomycetes on a root fragment of Perilla White Yellow-brown RF + 1 0 frutescene placed on HV agar. Total 18 18 (A) After two weeks of incubation at 30 C. (B) Scanning electron RA, rectinaculiaperti; RF, rectiﬂexibiles;S,spirales. micrograph of streptomycetes growing on the root fragment.

Table 3. Antimicrobial activity against plant pathogenic bacteria and fungi of streptomycetes isolated from the root surfaces of herbaceous plants and from the soil in which these plants were growing

No. of No. of active strains (%)y Isolation Streptomyces Agrobacterium Pseudomonas Alternaria Aspergillus Fusarium source strains rhizogenes syringae brassicicola niger oxysporum examined NBRC 14559 NBRC 3310 NBRC 31226 ATCC 9642 NBRC 31213 Root surface 71 48 (67.6%)a 55 (77.5%)a 42 (59.2%)a 57 (80.3%)a 54 (76.1%)a Soil 41 16 (39.0%)b 24 (58.5%)b 17 (41.5%)a 21 (51.2%)b 23 (56.0%)b Nine species of herbaceous plants were used (Brassica oleracea, Lupinus mutabilis, Mentha piperrita, Mentha suaveolens, Perilla frutescens, Pisum sativum, Solanum lycopersicum, Spinacia oleracea and Tagetes erecta). yWithin each column, active strain percentages with the same superscript are not signiﬁcantly diﬀerent (p < 0:05) according to the 2-test.

Eighteen streptomycete isolates, from each of the root tomycetes and soil streptomycetes. surface of P. frutescens and from soil sampled at a location The ability of the streptomycete isolates to inhibit 2 m distant from the growing plant, were selected at the growth of plant pathogenic bacteria and fungi was random and phenotypically grouped according to tradi- observed using an overlay method18) (Table 3). A signiﬁ- tional criteria, including aerial spore-mass color, substrate cantly (p < 0:05) higher percentage of organisms that were mycelium color, melanoid pigment production, and spore- active against these pathogens (except for Alternaria chain morphology.2) Table 2 shows that the 18 rhizoplane brassicicola NBRC 31226) was found for the streptomy- streptomycete isolates comprised 10 phenotypic groups. cetes isolated from root samples than for those isolated The 18 soil streptomycete isolates also comprised a number from the soil samples. of phenotypic groups. However, the number of strains in The MI&D method described in this paper makes use of each phenotypic group diﬀered between rhizoplane strep- the superior ability of streptomycete spores to withstand

68 ACTINOMYCETOLOGICA VOL. 21, NO. 2 desiccation. The resistance of aerial actinomycete spores to 7) Igarashi, Y.; T. Iida, T. Sasaki, N. Saito, R. Yoshida & T. desiccation has been well documented.19) Previous isolation Frumai: Isolation of actinomycetes from live plants and methods involved incubating root samples on appropriate evaluation of antiphytopathogenic activity of their metabo- agar media,6–10) but the root surface is colonized by lites. Actinomycetol. 16: 9–13, 2002. both streptomycetes and unicellular bacteria; therefore, it 8) Cao, L.; Z. Qiu, J. You, H. Tan & S. Zhou: Isolation and characterization of endophytic Streptomyces strains from is logical that undesirable unicellular bacteria could be surface-sterilized tomato (Lycopersicon esculentum) roots. eliminated using a simple drying method without adversely Lett. Appl. Microbiol. 39: 425–430, 2004. affecting the streptomycetes. The MI&D method is easy to 9) Hasegawa, S.; A. Meguro, M. Shimizu, T. Nishimura and H. carry out and results in the efficient isolation of diverse Kunoh: Endophytic actinomycetes and their interactions with rhizoplane streptomycetes that can be differentiated from host plants. Actinomycetol. 20: 72–81, 2006. soil-borne streptomycetes based on their taxonomic proper- 10) Okazaki, T.: Studies on actinomycetes isolated from plant ties and antimicrobial activities. Therefore, this method leaves. In Selective Isolation of Rare Actinomycetes (ed. I. could facilitate the isolation of novel strains that produce Kurtbo¨ke) pp. 102–122, Queensland, Queensland Complete useful antibiotics and other bioactive compounds. Several Printing Services, 2003. workers have suggested that rhizosphere streptomycetes 11) Birber, B.; J. Nuske, M. Ritzau & U. Grafe: Alnumycin, a play an important role in controlling the infection of new naphthoquinone antibiotic produced by an endophytic Streptomyces sp. J. Antibiot. 51: 381–382, 1998. roots by soil-borne pathogenic fungi and bacteria,2,3) so the 12) Igarashi, Y.; M. Ogawa, Y. Sato, N. Saito, R. Yoshida, H. present method could also be used to isolate actinomycete Kunoh, H. Onaka & T. Frumai: Fistupyrone, a novel inhibitor strains for use as biological control agents against phyto- of the infection of Chinese cabbage by Alternaria brassici- pathogens. cola, from Streptomyces sp. TP-A0569. J. Antibiot. 53: 1117–1122, 2000. REFERENCES 13) Castillo, U.; G. A. Strobel, E. J. Ford & 9 other authors: Munumbicins, wide-spectrum antibiotics produced by Strep- 1) Berdy, J.: Are actinomycetes exhausted as source of tomyces NRRL 30562, endophytic on Kennedia nigricans. secondary metabolites? In Proceedings of the 9th Interna- Microbiol. 148: 2675–2685, 2002. tional Symposium on the Biology of Actinomycetes (ed. 14) Hayakawa, M & H. Nonomura: Humic acid-vitamin agar, a V. G. Debabov, Y. V. Dudnik & V. N. Danilenko) pp. 13–34, new medium for the selective isolation of actinomycetes. J. Moscow, 1995. Ferment. Technol. 65: 501–509, 1987. 2) Williams, S. T.; M. Goodfellow & G. Alderson: Genus 15) Hayakawa, M.; T. Tamura, H. Iino & H. Nonomura: Pollen- Streptomyces Waksman and Henrici 1943. In Bergey’s baiting and drying method for the selective isolation of Manual of Systematic Bacteriology, vol. 4 (ed. S. T. Actinoplanes spp. from Soil. J. Ferment. Bioeng. 72: 433– Williams, M. E. Sharpe & J. P. Holt) pp. 2452–2492, 438, 1991. Baltimore, Williams and Wilkins, 1989. 16) Hayakawa, M.; Y. Yoshida & Y. Iimura: Selective isolation 3) Kutzner, H. J.: The family Streptomycetaceae. In The of bioactive soil actinomycetes belonging to the Strepto- Prokaryotes (ed. M. P. Starr, H. Stolp, H. G. Tru¨per, myces violaceusniger phenotypic cluster. J. Appl. Microbiol. A. Balows and H. G. Schlengel) pp. 2028–2090, Berlin, 96: 973–981, 2004. Springer Verlag, 1981. 17) Hasegawa, T.; M. Takizawa & S. Tanida: A rapid analysis 4) Coombs J. T. & C. M. M. Franco: Isolation and identification for chemical grouping of aerobic actinomycetes. J. Gen. of actinobacteria from surface-sterilized wheat roots. Appl. Appl. Microbiol. 29: 319–322, 1983. Environ. Microbiol. 69: 5603–5608, 2003. 18) Otoguro, M.; M. Hayakawa, T. Yamazaki & Y. Iimura: An 5) Hunter, J. C.; M. Fonda, L. Sotos, B. Toso & A. Belt: integrated method for the enrichment and selective isolation Ecological approaches to isolation. Dev. Ind. Microbiol. 25: of Actinokineospora spp. in soil and plant litter. J. Appl. 247–266, 1984. Microbiol. 91: 118–130, 2001. 6) Sardi, P.; M. Sarracchi, S. Quaroni, B. Petrolini, G. E. 19) Kalakoutskii, L. V. & L. M. Pouzharitskaja: The streptomy- Borgonori & S. Merli: Isolation of endophytic Strepto- ces spore: its distinct feature and germinal behaviour. In myces strains from surface-sterilized roots. Appl. Environ. Actinomycetales (ed. G. Sykes & F. A. Skinner) pp. 155–171, Microbiol. 58: 2691–2693, 1992. London, Academic Press, 1973.